Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids; hybridization involves base-pairing between complementary nucleic acid strands. When single-stranded nucleic acids are used as probes to identify specific target sequences of nucleic acids, probes of known sequences are exposed to and incubated in sample solutions containing sequences to be identified. If a sequence hybridizes to a probe of a known sequence, the sequence is necessarily the specific target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and thus form double-stranded stable molecules, and a variety of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
Hybridization of surface-bound probes to solution-based targets is an effective means to analyze a large number of DNA or RNA molecules in parallel. Specific probes of known sequences are attached to the surface of a solid substrate in known locations. The probes are usually immobilized on a solid support having a surface area of typically less than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of probes. A mobile-phase sample containing labeled targets, e.g., a buffered aqueous solution containing target DNA, is contacted with and allowed to react with the surface. By detecting the labels to determine whether hybridization has occurred at specific locations, it is possible to determine the composition of the sample and the sequences of the unknown targets. Alternatively, target biomolecules may be bound to the surface while labeled probes are contained in the mobile phase. In either case, the hybridization reaction typically takes place over a time period that can be many hours, for a typical sample containing target material in the concentration range in the picomolar domain.
In the preparation of arrays such as those for use in nucleic acid hybridization, reagents may be applied to predetermined locations on the surface of a substrate. Generally, a surface is first cleaned or otherwise prepared by exposure to a fluid containing a reagent. Then, array preparation will involve application of biomolecule-containing fluids at discrete locations. For nucleic-acid probe array preparation, the biomolecule-containing fluid may contain the already-formed probes that can bind with the surface, or a specific nucleotide that will later constitute a portion of a probe that is synthesized in situ on the surface. Then, treatment of a portion of or the entire surface with a different fluid may follow. The steps may be repeated a number of times in situ to prepare the desired array. Once an array of probes is formed on a substrate surface for hybridization with target molecules in a sample fluid, hybridization may be carried out by uniformly exposing the entire substrate surface to the sample fluid.
It is apparent, then, that surface coating by a fluid is an important aspect in array technology, particularly in the field of biomolecular arrays. Important aspects of coating procedures include the amount of fluid used and the rate of throughput. In general, coating procedures should employ only a small quantity of fluid, for a number of reasons. First, the fluid may contain expensive or rare reagents, and waste of such fluids is undesirable. Second, many ordinary reagents that are used in array preparation are toxic, and decreasing their use is desirable in order to lower the risk of human exposure. A high throughput rate also implies that it takes less time to coat each substrate surface, thereby also lowering the risk of human exposure during the coating procedure.
Another important aspect of coating procedures is uniformity of coverage. For biomolecular arrays, it is desirable to uniformly apply a fluid onto a substrate surface to ensure that each feature is attached or formed under similar conditions. In addition, during use of a formed array containing surface-bound probes, uniform distribution of sample fluid to ensure proper hybridization is necessary. Without uniform fluid distribution, resultant hybridization data will be compromised.
One method by which a surface may be coated with a small amount of fluid is through the use of a flow cell assembly. Variations on the use of a flow cell are described in U.S. Pat. Nos. 4,596,695 to Cottingham and 5,145,784 to Cox et al. The basic flow cell method typically provides that a cover and substrate are positioned parallel to each other. A gap is thus formed between the cover and the substrate. To control the size of the gap, one or more spacers having a selected height are disposed within the gap. In addition, the cover, the spacers and the substrate are arranged such that a chamber is provided having an inlet channel and an outlet channel. By creating an appropriate pressure gradient between the inlet and outlet channels, fluid fills the chamber by laminar flow, coating the surface of the substrate within the chamber. By controlling the volume in the chamber through the proper selection of the spacer height, the amount of fluid needed to coat the surface can be reduced.
The use of the flow cell method has a number of drawbacks. First, uniform coating requires laminar flow of the fluid. Laminar flow regime generally implies that there is an absolute upper limit to the volumetric rate given the geometry of the chamber. Second, to increase the flow rate of the fluid, the pressure gradient between the inlet and outlet channels must be increased. However, pressure surges that are generated while increasing the pressure gradient tend to cause the flow cell assembly to leak, either at the cover/support interface or at the support/substrate interface. Third, any irregularity in the surface profile of the substrate tends to disrupt laminar flow. As a result, air pockets may be formed and trapped within the flow cell assembly that will interrupt contact between the fluid and the substrate. Thus, while the use of a flow cell tends to lower the amount of reagent fluid waste, the gain in lowered waste is offset by diminishing throughput.
Spin coating may be employed to quickly and uniformly coat a fluid on a substrate. Spin coating is usually performed by dispensing the fluid at or near the center of a substrate. The substrate is spun either during or after the reagent is dispensed such that the fluid spreads radially and outwardly to cover the entire substrate. In this method, the volume needed to cover a surface depends on fluid property, e.g., viscosity and surface tension, and the surface energy of the substrate. When a low energy surface is provided, a relatively large volume fluid is needed to cover the entire surface. Without sufficient volume, applied fluid tends to exhibit clustering behavior and does not cover the entire surface uniformly. Thus, a relatively large amount of fluid is first applied to the surface at a low spin speed to cover the entire surface. Then, the substrate is spun at a higher speed to remove excess fluid. Consequently, while spin coating may be advantageous in terms of high throughput, it is a relatively wasteful technique.
Thus, there is a need to provide a method and apparatus to coat a substrate surface with a small volume of fluid quickly and uniformly without relying on spin coating or an ordinary flow cell.